Abstract

Vaults are 13 megadalton ribonucleoprotein particles composed
largely of the major vault protein (MVP) and two high molecular weight
proteins, p240 and p193, and a small vault RNA (vRNA). Increased levels
of MVP expression, vault-associated vRNA, and vaults have been linked
directly to multidrug resistance (MDR). To further define the putative
role of vaults in MDR, we produced monoclonal antibodies against the
Mr 193,000 vault protein and studied its
expression levels in various multidrug-resistant cell lines. We find
that, like MVP, p193 mRNA and protein levels are increased in various
multidrug-resistant cell lines. Subcellular fractionation of vault
particles revealed that vault-associated p193 levels are increased in
multidrug-resistant cells as compared with the parental, drug-sensitive
cells. Furthermore, protein analysis of postnuclear supernatants and
coimmunoprecipitation studies show that drug-sensitive MVP-transfected
tumor cells lack this up-regulation in vault-associated p193. Our
observations indicate that vault formation is limited not only by the
expression of the MVP but also by the expression or assembly of at
least one of the other vault proteins.

INTRODUCTION

Resistance to a broad range of cytostatic drugs
(MDR3
) can be mediated by Pgp or the MRP1. Pgp and MRP1 function as efflux
pumps, decreasing intracellular drug accumulation (1)
. In
clinical drug resistance, however, other mechanisms may play a role,
e.g., involving drug sequestration into exocytotic vesicles.
Evidence has been obtained that subcellular particles named vaults may
play a critical role in such a mechanism (2, 3)
.

Vaults are 13 megadalton ribonucleoprotein particles
containing three proteins of Mr
240,000, 193,000, and 110,000, respectively, and a small untranslated
vRNA. A vault interacting protein of
Mr 54,000 is occasionally observed in
rat liver vault preparations (2)
. Vaults are widely
distributed throughout eukaryotes, and their morphology is highly
conserved among various species. The remarkable structural conservation
and broad distribution of vaults suggest that their function is
essential to eukaryotic organisms and that the structure of the
particle must be important for its function (4, 5, 6)
.
Although vault function is undetermined, it has been proposed that
vaults may mediate transport of various substrates (7, 8)
.
A role for vaults in intracellular traffic might be mediated by binding
to cellular organelles through direct interaction with its targets
(9)
. Recently, an interaction of vaults with intracellular
steroid hormone receptors has been reported (10)
. Although
the majority of vault particles are distributed throughout the cytosol,
a portion of vaults has been localized to the nuclear membrane at
or near the nuclear pore complex. Furthermore, the recent
three-dimensional reconstruction of the vault particle reveals a hollow
interior, which may prove important in the transport/sequestration of
large substrates (11)
. On the basis of striking
similarities between vault particle mass and symmetry and the
predictive mass of the putative central plug of the nuclear pore
complexes, a role of vaults in nucleocytoplasmic exchange has been
proposed (12)
.

The discovery of a key role of VR-MDR in clinical drug
resistance depended on the molecular identification of the LRP as the
human MVP (3)
. LRP had been first identified in a
non-small cell lung cancer cell line, selected in vitro for
DOX resistance. The protein was subsequently found to be overexpressed
in many human tumor cell lines characterized by their MDR phenotype, in
the absence of drug accumulation defects such as mediated by Pgp
(13)
. Moreover, LRP expression closely reflected known
chemoresistance characteristics in broad panels of unselected tumor
cell lines and untreated clinical cancers of different histogenetic
origins (14, 15)
. Results from several, but not all,
clinicopathological studies showed that LRP expression at diagnosis,
rather than Pgp or MRP1 expression, is a strong and independent
prognostic factor for poor response to chemotherapy and/or outcome,
e.g., in ovarian carcinoma and leukemias (16)
.
Most importantly, Kitazono et al.(17)
demonstrated recently, using a LRP induction system and LRP-specific
ribozymes, that LRP is involved in resistance to Adriamycin,
vincristine, VP-16, Taxol, and gramicidine D and has an important role
in the transport of Adriamycin between the nucleus and the cytoplasm in
the SW-620 human colon carcinoma cell line. To avoid confusion, we will
hereafter refer to LRP as MVP.

Studies on the role of vaults in MDR, including the cloning of
the MVP cDNA, have thus far been based on polyclonal
antisera and two mAbs, LRP-56 and LMR-5 (13, 18)
, directed
against the MVP. To further define the role of vaults in MDR, the other
components must be characterized. The human vRNA
genes have been cloned, and within tumor cells, not all of the
vRNA was found to be vault- associated. Sedimentation measurements of
vault components in VR-MDR cells have revealed up to a 15-fold increase
in vault copy number, coupled with a comparable shift of vRNA to the
100,000 × g pellet, demonstrating that vault
formation is limited by expression of MVP and/or one of the other
vault proteins (19)
. Because MVP-transfected cells did not
show a drug-resistant phenotype (3)
, the other vault
components are thought to be essential for vaults to play a role in
MDR.

Here we describe the production of the first mAbs against the
Mr 193,000 vault protein. The p193 was
recently identified by its interaction with the MVP in a yeast
two-hybrid screen, and its identity was confirmed by peptide sequence
analysis (20)
. Results from protein analysis of
postnuclear supernatants and subcellular fractions, Northern analysis,
immunocytochemical and coimmunoprecipitation studies show that:
(a) p193 and MVP are both increased in various MDR cell
lines and; and (b) vault-associated p193 levels are
up-regulated in these MDR cell lines but not in a drug-sensitive,
MVP full-length, cDNA-transfected cell line, supporting the
conclusion that functional vault formation is limited by expression of
MVP and expression of at least one of the other vault components, p193.

Screening, Cloning, and Isotyping.

After 9 days of growth in selective medium, the hybridoma
supernatants were tested for the presence of antibodies of interest by
ELISA. Plates (96-well) were coated with 5 μg/ml of the immunization
antigen or, as a control, 5 μg/ml of pET-28a(+) transformed E.
coli BL21 (DE3) bacterial lysate induced to produce a His-tagged,β
-galactosidase recombinant protein. Hybridomas secreting antibodies
of interest were subcloned three times by limiting dilution.
Immunoglobulin subtypes of the selected mAbs were determined using an
isotype reagent kit (Boehringer Mannheim, Indianapolis, IN).

PEPSCAN.

All overlapping dodecapeptides (12-mers) covering amino acids
408–611 of the p193 protein (beginning with the 12-mers 408–419,
409–420, and so forth) were synthesized and screened using the
minipepscan method as described previously (24, 25)
. In
credit card format of mini-PEPSCAN cards (455 peptides/card), the
binding of the anti-p193 mAbs to each peptide was tested in a
PEPSCAN-based ELISA. The 455-well credit card format polyethylene
cards, containing the covalently linked peptides, were incubated with
mAbs p193-4, p193-6, and p193-10 (8, 15, and 10 μg/ml, respectively).
After washing, the cards were incubated with rabbit antimouse
peroxidase (Dako, Glostrup, Denmark; 1 h at 25°C), and
subsequently, the peroxidase substrate
2,2′-azino-di-3-ethylbenzthiazoline sulfonate and 2 μl/ml 3%
H2O2, was added. The color
development of the ELISA was measured after 1 h and quantified
with a CCD camera and an image processing system. The setup consisted
of a CCD camera and a 55-mm lens (Sony CCD Video Camera XC-77RR; Nikon
Micro-Nikkor 55-mm f/2.8 lens), a camera adaptor (Sony Camera adaptor
DC-77RR), and the Image Processing Software package TIM, version 3.36
(Difa Measuring Systems, Breda, the Netherlands).

MVP Antibodies.

MVP expression was studied using the rabbit polyclonal
antibody Pab W. Pab W was raised as follows. A
NcoI-EcoRV fragment of 2631 bp (amino acids
1–871) was cloned between the NcoI and filled-in
HindIII sites of the pGEX-KG polylinker (26)
.
The resulting glutathione S-transferase MVP fusion protein
was synthesized in E. coli DH5α as described by Guan and
Dixon (26)
, except that the cells were lysed by
sonication. The largely soluble glutathione S-transferase
MVP fusion protein was bound to glutathione-Sepharose 4B beads
(Pharmacia Biotechnology, Uppsala, Sweden), after which the MVP part
was released by a thrombin digest. The fraction containing the
MVP was concentrated by freeze-drying and used to immunize a
rabbit. Also, MVP expression was studied with two MVP-specific murine
mAbs (both of the IgG2b subclass) obtained in our laboratory: LRP-56,
which was raised by immunization of mice with the MDR tumor cell line
SW-1573/2R120 (13)
; and MVP-37, raised in mice against the
above-described MVP construct.

Immunocytochemistry.

Cytocentrifuge preparations of tumor cell lines were
air-dried, fixed at room temperature in acetone for 10 min or 3% (w/v)
paraformaldehyde/0.4% (w/v) glucose in PBS for 10 min. The
paraformaldehyde-fixed cells were washed two times with PBS and then
incubated with 20 mm glycine (pH 7.5) in PBS for 10 min to
block unreacted aldehyde groups. This was followed by two washes in
PBS/0.2% (w/v) BSA. Denaturation of intracellular proteins was done by
applying 50 μl of 6 n guanidine hydrochloride in 50
mm Tris-HCl (pH 7.5) to the cytospin preparations for 10
min (27)
. The cells were then rinsed three times with
PBS/0.2% BSA. All antibody dilutions were made in PBS/1% BSA. Between
incubation steps, the acetone-fixed cytospin preparations were washed
(three times during 15 min) with PBS; paraformaldehyde-fixed
preparations were washed with PBS/0.2% BSA. Primary antibodies were
applied for 60 min at room temperature to the acetone-fixed cytospin
preparations and for 30 min at 37°C to the paraformaldehyde-fixed
preparations. Irrelevant mouse IgG1 (Cappel, Organon Teknika Aurora,
OH) was used as negative control. Subsequently, the preparations
were incubated with biotinylated rabbit antimouse
F(ab′)2 fragments (Dako; 60 min at room
temperature), followed by peroxidase-conjugated streptavidin (Zymed,
San Francisco, CA; 30 min at room temperature). Bound peroxidase was
visualized with 4 mg (w/v) amino-ethyl-carbazole and 0.02% (v/v)
H2O2 in 0.1 m
NaAc (pH 5.0), nuclei were counterstained with hematoxylin, and the
cytospin preparations were mounted with Kaiser’s mounting medium.

Northern Analysis.

Total RNA was isolated following the procedure of Chomczynski
and Sacchi (30)
. The RNA (20 μg) was fractionated on a
formaldehyde-agarose gel and transferred to Hybond-N membrane (Amersham
Corp., Little Chalfont, United Kingdom). Hybridization was carried out
according to the manufacturer’s recommendation with a randomly primed
p193 probe (bases 4515–5490). Hybridized bands were visualized on a
Phosphor- Imager screen (Molecular Dynamics).

Protein Analysis of Postnuclear Supernatant.

Extracts were prepared from various drug-sensitive, resistant,
and revertant cell lines by the following procedure. Cells were
harvested and resuspended in cold buffer A [50 mm Tris-Cl
(pH 7.4), 1.5 mm MgCl2, and 75
mm NaCl] containing 0.5% NP40 and 1 mm
phenylmethylsulfonyl fluoride. All subsequent steps were performed at
4°C. Samples were vortexed, incubated on ice for 5 min, and
centrifuged at 9000 × g for 20 min. The
resulting supernatant was designated as the postnuclear supernatant.
Protein concentration was determined with a Bio-Rad protein assay
(Bio-Rad, Richmond, CA). Protein samples were fractionated by SDS/6%
PAGE and subsequently transferred to nitrocellulose filter by
electroblotting. After blotting, the filters were blocked for at least
2 h in block buffer (PBS containing 1% BSA, 1% milk powder, and
0.05% Tween 20), followed by overnight incubation with the primary
antibodies in block buffer/10% FCS. Immunoreactivity was visualized
with peroxidase-conjugated swine antirabbit or rabbit antimouse
immunoglobulins (Dako) in block buffer/10% FCS, followed by staining
with 0.05% chloronaphtol and 0.03%
H2O2 in PBS. Protein levels
were determined by densitometric scanning (GelDoc; Bio-Rad) of the
filters. The density of the protein bands was analyzed using the
software of the manufacturer (Molecular Analyst; Bio-Rad). The values
obtained were expressed as absorbance (A) × mm2.

Subcellular Fractionation.

Nuclear, S100, and P100 extracts were prepared from various
drug-sensitive, resistant, and revertant cell lines as described
previously (19)
, except resuspended at a concentration of
4 × 107 cell/ml. Equal volume
amounts of fractions were analyzed for protein content. Protein samples
were solubilized in SDS sample loading buffer, fractionated on 7.5%
SDS-PAGE, and transferred to Hybond-C (Amersham Corp.) by
electroblotting. Western blots were performed using the mouse anti-p193
mAbs following established procedures. Reactive bands were detected
using the enhanced chemiluminescence system (Amersham Corp.).

Immunoprecipitation.

A2780, AC16, and GLC4/ADR cells were used in the
immunoprecipitation assays. Aliquots of postnuclear supernatants
containing 750 μg of protein (prepared as described in “Protein
Analysis of Postnuclear Supernatant”) were brought up to 500 μl and
incubated for at least 2 h at 4°C with 8 μg of mAb.
Antibody-antigen complexes were recovered by incubation with 14% w/v
protein A-Sepharose CL-4B (Pharmacia Biotech BV, Woerden, the
Netherlands). Precipitated proteins were detected by immunoblotting (as
described above in “Protein Analysis of Postnuclear Supernatant”).

Electron Microscopy.

Negative staining was performed by adsorbing
immunoprecipitates of the GLC4/ADR cells onto Formvar-coated 75-mesh
copper grids (Stork Veco, Eerbeek, the Netherlands) for 5 min, blotting
of the sample, and adding 1% uranyl acetate for 4–5 min. Excess stain
was than removed by blotting, and the specimens were air dried and
viewed on a Jeol 1200 EX electron microscope.

RESULTS

Generation of mAbs against p193.

Using popliteal lymph nodes from mice immunized with an
E. coli lysate transformed with the pET28a(+) expression
vector containing amino acids 408–611 of the p193 cDNA, murine
hybridomas were generated and screened for their ability to detect the
immunization antigen by ELISA. Three stable, cloned hybridoma cell
lines, designated p193-4, p193-6, and p193-10 were obtained. mAb p193-4
was determined to be of the IgG1κ subclass, mAb p193-6 of the
IgG2bκ subclass, and mAb p193-10 was an IgG2aκ. PEPSCAN analysis
showed that the smallest peptide sequence recognized by mAb p193-4 is
HPGE (amino acids 491–494), by mAb p193–6 FSKVEDY (amino acids
593–599), and by mAb p193-10 VALGK (amino acids 506–510; Fig. 1⇓
).

Coordinate expression of p193 and MVP in tumor cells.
Western analysis of p193 (using the mAb p193-4, ∼15 μg/ml) and MVP
(using the Pab W, 1:500) levels in 80 μg of postnuclear supernatants
from several MDR cell lines, including the parental cell lines.

Northern analysis of total RNA from the parental GLC4 cell
line and its drug-resistant, derivative cell line GLC4/ADR revealed
that p193 mRNA levels are increased in the GLC4/ADR cell line, in
conjunction with the observed increased expression of p193 protein. In
the drug revertant GLC4/REV line, which was isolated by culturing the
cells in the absence of drug, resulting in a much less drug-resistant
cell line, the p193 mRNA level was intermediate (Fig. 3)⇓
. Thus, both p193 mRNA and protein expression are regulated according
to drug resistance level, concomitant with MVP mRNA and protein levels
(3, 19, 31)
.

Immunocytochemical staining of cytocentrifuge preparations
confirmed the correlation between p193 and MVP expression in the tumor
cell lines. Although less intense, the granular p193 staining
throughout the cytoplasm of drug-resistant GLC4/ADR (Fig. 4b)⇓
and SW-1573/2R120 cells (Fig. 4d)⇓
is similar to
that observed by MVP staining (Fig. 4, f and h⇓
,
respectively). No expression of p193 was detected in the parental
drug-sensitive GLC4/S (Fig. 4a)⇓
and SW-1573 (Fig. 4c)⇓
cell lines, which show no and only weak expression of
MVP (Fig. 4, e and g⇓
, respectively).

Overlapping Distribution of p193 and MVP in Tumor Cell Lines.

By double immunofluorescence labeling experiments, we compared
the subcellular localization of the p193 protein with that of the MVP
protein. Double labeling of vault-overexpressing GLC4/ADR cells using
mAb p193-4 against p193 and mAb MVP-37 against MVP revealed an
identical cytoplasmic granular staining pattern of both vault proteins
(Fig. 5, a–c)⇓
. Similarly, colocalization of p193 and MVP
distribution was found in the vault-positive SW-1573/2R120 cells (data
not shown). As described previously (13)
, only 1–3% of
the cells from the SW-1573/2R160 cell line stained positive for MVP
(Fig. 5e)⇓
. These cells also showed p193 (Fig. 5d)⇓
staining, which completely colocalized with the MVP (Fig. 5f)⇓
. No R-phycoerythrin/p193 labeling was
observed when irrelevant mouse IgG1 (Cappel) replaced p193-4. No
FITC/MVP labeling was observed when irrelevant mouse IgG2b
(anti-chromogranin A; Dako) replaced MVP-37.

Up-Regulation of Vault-associated p193 in MDR Tumor Cells.

By subcellular fractionation, Kedersha and Rome
(2)
showed that vaults pellet at 100,000 × g (P100), and that all of the MVP is associated with this
fraction and is assembled into vaults. In contrast, only a portion of
the total cellular vRNA fractionates to the P100, where it is
associated with vaults. The non-vault-associated vRNA fractionates in
the soluble or S100 fraction (19)
. Here, we determined the
p193 levels in these fractions of the parental, drug-sensitive GLC4
cell line and its derivative cell lines, the drug-resistant GLC4/ADR
and drug revertant GLC4/REV (Fig. 6)⇓
. Unlike the MVP protein, the p193 protein was observed in all of the
fractions: N, S100, and P100; however, the greater part fractionated
with the P100. The detection of p193 in the S100 fraction indicates
that besides vault-associated p193 also non-vault-associated p193 is
present. Furthermore, a comparison of parental, resistant, and
revertant cells revealed that a higher amount of p193 fractionates with
the P100 of the MDR GLC4/ADR cell line. Although less obvious, the N
and S100 fractions of these drug-resistant cells also contained
increased levels of p193 protein as compared with the parental and
revertant cells. Analysis of additional MDR lines, including the
parental, drug-sensitive lines (SW-1573, SW-1573/2R120, MCF-7, and
MCF-7/MR; data not shown) confirmed our observation that in MDR cells
clearly a higher amount of the p193 is localized in the P100, the
fraction that reflects the level of assembled vault particles.

Vault-associated p193 (P100) levels are up-regulated in
GLC4/ADR cells. p193 levels were determined by Western blotting (using
mAb p193-4 ∼20 μg/ml) in subcellular fractions as described in“
Materials and Methods.” Sample extracts for each cell line are in
groups of three: nuclear (N), high-speed supernatant
(S), and high-speed pellet (P) from the
parental GLC4, resistant GLC4/ADR, and revertant GLC4/REV.

To further analyze the association of the p193 with assembled
vault particles, we immunoisolated vault particles from the postnuclear
supernatant of the multidrug-resistant GLC4/ADR cell line using the
anti-MVP mAb LRP-56. The precipitated structures were negatively
stained and examined by electron microscopy. The ovoid structures that
were revealed have similar dimensions to those seen in purified vault
samples described previously (Fig. 7⇓
; Refs. 2,, 4, 5, 6
). The slight distortion of the vault
morphology is probably attributable to the presence of the bound
antibodies on the surface of the vault particle or disruption of the
particle from antibody interactions. No vault structures were detected
in control immunoprecipitates (not shown). Immunoprecipitation of the
MVP/vaults from postnuclear supernatant of the multidrug-resistant
GLC4/ADR cell line, followed by detection of MVP as well as p193 by
immunoblotting experiments, showed clear coimmunoprecipitation of the
p193 with the MVP (Fig. 8)⇓
. Control immunoprecipitation with an isotypic mAb revealed neither MVP
nor coimmunoprecipitation of p193. Both proteins were localized in the
corresponding supernatant. In contrast with our fractionation data, no
soluble p193 was detected in the supernatant that remained after MVP
precipitation. This is not surprising because the supernatants that
were examined are much less concentrated than the immunoprecipitation
samples. As a result, the relatively low levels of non-vault-associated
p193 may remain below the level of detection.

Coimmunoprecipitation of p193 with MVP in
multidrug-resistant GLC4/ADR cells, not in drug-sensitive AC16 and
parental A2780 cells. Western analysis of p193 (using the mAb p193-4∼
15 μg/ml) and MVP (using the Pab W 1:500) in MVP
immunoprecipitates (using the mAb LRP-56) and isotypic, control
immunoprecipitates and the supernatants of these immunoprecipitates.
The precipitations were carried out on postnuclear supernatants of the
cell lines A2780, AC16, and GLC4/ADR.

Lack of p193 Expression in MVP-transfected,
Drug-sensitive Cells.

We have shown previously by transfection studies that
overexpression of the MVP cDNA alone is not sufficient to confer a
drug-resistant phenotype. Because vaults are multisubunit particles, we
reasoned that additional components could be required for functional
vault formation (3)
. To examine the possibility that the
assembly of functional vault particles is limited by p193 expression,
we determined the level of p193 expression in the
MVP-transfectant cell line AC16, which was derived from the
parental, drug-sensitive A2780 ovarian carcinoma cell line
(3)
. The GLC4/ADR cell line was included as a positive
control. As analyzed by Western blotting, the AC16 cells clearly
overexpress MVP as compared with the parental A2780 cells. However, a
comparison of the p193 expression levels in the MVP-transfected and the
parental cell line revealed no concomitant induction of p193 expression
in the MVP transfectant (Fig. 9)⇓
. Furthermore, immunoprecipitation of the MVP from the postnuclear
supernatant of the AC16, followed by SDS-PAGE analysis, showed only
detection of MVP (Fig. 8)⇓
.

DISCUSSION

There is still much speculation about the function of vaults. As
an apparently ubiquitous and highly conserved subcellular particle
(4, 5, 6,, 8)
, vaults must have an important function in
fundamental cell processes. On the basis of structure analyses and
subcellular localization studies, it has been postulated that they
mediate nucleocytoplasmic exchange as well as vesicular transport of
various substrates, including cytostatic drugs. In support of this
view, the clinical relevance of vaults in the prediction of a
multidrug-resistant phenotype in numerous cancer cell types is well
documented (14, 15, 16)
. Recently, Kitazono et al.(17)
showed that reduction of MVP expression by use of
MVP-specific ribozymes in a cell line induced to overexpress MVP by
exposing cells to sodium butyrate was enough to prevent drug
resistance. Furthermore, the nuclei isolated from sodium butyrate
untreated cells or those from treated cells in the presence of anti-MVP
antibodies accumulated cytostatic drugs, but those from treated cells
in the absence of antibodies did not. These findings provide the first
causal relationship between MVP expression and drug resistance.

Studies on the function and structure of vaults to date have
been focused on the MVP and vRNA. In this study, we generated mAbs
against the p193 and used these to further characterize the p193 in
human tumor cell lines. A strong positive correlation was found between
p193 and MVP expression by Western analysis, suggesting that besides
MVP also p193 expression is indicative for VR-MDR. In addition,
analysis of total RNA indicated that besides the p193 protein also p193
mRNA levels increase accordingly to an increase in the number of
vaults. Immunocytochemical analysis showed that the distribution of
p193 staining is compatible with the cytoplasmic location of
MVP/vaults. Paraformaldehyde fixation followed by a guanidine
hydrochloride denaturation pretreatment (27)
is a
prerequisite for exposing the epitopes recognized by the anti-p193
mAbs. This protocol could not retrieve the antigenic site for the
anti-MVP mAb LRP-56, which gives a positive signal in cells fixed with
acetone. This is not surprising because the anti-p193 mAbs were raised
against a fusion protein and therefore are more likely to be unreactive
with their antigens in a more native conformation than the anti-MVP mAb
LRP-56, which was raised against tumor cell lysate.

Comparison of the distribution of the p193 with the MVP
protein in tumor cells reported earlier to contain high amounts of
vault particles (19)
reveals an identical staining pattern
in corresponding immunofluorescent images. Typical vault granules are
present in the entire cytoplasm, which colocalize in the double-exposed
image. The double staining results strongly suggest a high degree of
association of the p193 with MVP/vaults. On the basis of MVP
overexpression, vaults have been reported to be most abundant in
epithelial cells (5, 15)
. Although the precise tissue
distribution of the p193 is still under investigation, we found p193 to
be present in normal human lung, with highest expression in the
epithelial cells lining the respiratory tract. Thus, vault expression
in normal human tissues also involves p193 expression.

Upon subcellular fractionation, the MVP is exclusively present
in the large vault complex retained in a particulate fraction (2, 5, 19, 32)
. Western analysis of similar subcellular fractions of
tumor cell lines demonstrates the presence of vault-associated as well
as a relatively low level of non-vault-associated p193. In a previously
reported study, (20)
the presence of non-vault-associated
p193 was also observed in the cytosol and nucleus by Western blot
analysis upon subcellular fractionation. In contrast with the present
findings, the nuclear localization of p193 was also shown in these
studies by immunofluorescence using an anti-p193 polyclonal antibody.
Probably the relatively low level of non-vault-associated p193 in the
drug-resistant cell lines remains below the detection level in our
immunofluorescence double-labeling experiments using anti-p193 mAbs, or
the soluble p193 signal is masked by the abundance of MVP located in
the cytoplasm. A comparison upon subcellular fractionation of resistant
and parental tumor cell lines reveals a clear increase in
vault-associated p193 levels in the resistant cells. Furthermore, in
the revertant GLC4 cell line, which was isolated by culturing in the
absence of drug but still is a drug-resistant cell line (albeit at a
lower concentration of drug), vault-associated p193 protein levels
decrease. Thus, an increase in the general pool of vaults (as in the
drug resistant cells) results in an increase in vault-associated p193.

To further evaluate the association of p193 with MVP/vaults,
we performed immunoisolation of vault particles from GLC4/ADR cells.
Negative staining electron microscopy showed that vault particles were
isolated as judged by the structure resemblance to vaults described
previously (2, 5, 6)
. Western analysis of the
immunoisolates revealed coimmunoprecipitation of the p193 with the MVP,
demonstrating the specificity of p193 association with MVP/vaults.

Consistently, vault formation is limited by the expression of
MVP (19)
. However, the previously constructed
MVP transfectant tumor cell line AC16 shows no signs of drug
resistance (3)
. This is not unexpected because the MVP
comprises only 70% of the vault particle. Therefore, additional
components of the vault particle could also be required for vault
function and drug resistance. Within tumor cells, vRNA was found to be
in considerable excess to MVP (19)
, suggesting that
in the AC16 cells, this component is not limiting functional vault
formation. Western analysis revealed that endogenous p193 was not
overexpressed. Furthermore, no detectable coimmunoprecipitation of p193
with MVP was found in the immunoisolates of the MVP transfectant AC16.
These findings support our view that MVP transfection did
not result in an acquired VR-MDR phenotype, because the assembly of
functional vault particles is impaired by the lack of overexpression of
at least another vault constituent and subsequent vault association,
leading to high numbers of incomplete dysfunctional vault particles.

Taken together, using newly developed mAbs against the p193,
we have demonstrated that the MVP and p193 are co-up-regulated in
various MDR cell lines. Furthermore, we present evidence that
functional vault formation is not only limited by expression of the MVP
but also by expression of the p193. Considering that the expression of
two vault proteins is essential for functional vault formation, the
contribution of the Mr 240,000
vault protein, which was recently identified as a component in the
telomerase complex (telomerase-associated protein, TEP1; Ref.
33
) needs to be clarified to complete the picture on vault
function and the role of VR-MDR in clinical drug resistance.

Acknowledgments

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.